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SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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CD38 Expression in Human Germ-Cell-Related Cells and Epigenetic Changes in hPGCLCs(A) FACS analysis of NANOS3-mCherry and CD38 on WIS2-NANOS3-mCherry cell line cultured in 4i medium and on day 4 and 5 embryoids following hPGCLC induction. Ratios of CD38 low and high expression in the NANOS3-mCherry-positive cells are indicated.(B) FACS histogram of CD38 low and high populations in TCam-2.(C) FACS analysis of CD38 and TNAP on genital ridges isolated from a week 6 human embryo.(D) Expression analysis by RT-qPCR for FACS-sorted TNAP-positive 4i hESCs (TNAP+ hESC) and CD38 low or high/NANOS3-mCherry day 5 hPGCLCs. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(E and F) Immunofluorescence analysis for 5hmC (E) and TET1 (F) on day 4 embryoids cryosection. OCT4 or BLIMP1 were used to identify hPGCLCs (highlighted). Scale bars, 50 μm.(G) Quantification of immunofluorescence intensity of various epigenetic marks/modifiers in hPGCLCs and somatic neighbors in day 1–4 embryoids (see also Figures S3A–S3C). For UHRF1, only KI-67-positive (proliferating) cells were used for quantification. Numbers below each box denotes number of cells analyzed. Black central line represents the median, boxes and whiskers represent the 25th and 75th, and 2.5th and 97.5th percentiles, respectively. Wilcoxon signed-rank test was used to test for statistical significance. #p < 0.05; ∗p < 0.0001.
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fig3: CD38 Expression in Human Germ-Cell-Related Cells and Epigenetic Changes in hPGCLCs(A) FACS analysis of NANOS3-mCherry and CD38 on WIS2-NANOS3-mCherry cell line cultured in 4i medium and on day 4 and 5 embryoids following hPGCLC induction. Ratios of CD38 low and high expression in the NANOS3-mCherry-positive cells are indicated.(B) FACS histogram of CD38 low and high populations in TCam-2.(C) FACS analysis of CD38 and TNAP on genital ridges isolated from a week 6 human embryo.(D) Expression analysis by RT-qPCR for FACS-sorted TNAP-positive 4i hESCs (TNAP+ hESC) and CD38 low or high/NANOS3-mCherry day 5 hPGCLCs. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(E and F) Immunofluorescence analysis for 5hmC (E) and TET1 (F) on day 4 embryoids cryosection. OCT4 or BLIMP1 were used to identify hPGCLCs (highlighted). Scale bars, 50 μm.(G) Quantification of immunofluorescence intensity of various epigenetic marks/modifiers in hPGCLCs and somatic neighbors in day 1–4 embryoids (see also Figures S3A–S3C). For UHRF1, only KI-67-positive (proliferating) cells were used for quantification. Numbers below each box denotes number of cells analyzed. Black central line represents the median, boxes and whiskers represent the 25th and 75th, and 2.5th and 97.5th percentiles, respectively. Wilcoxon signed-rank test was used to test for statistical significance. #p < 0.05; ∗p < 0.0001.

Mentions: A heat map of mRNA expression revealed that hPGCLCs and gonadal hPGCs shared expression of early PGCs (BLIMP1, TFAP2C, DND1, NANOS3, UTF1, ITGB3, and KIT) and pluripotency genes (TNAP, OCT4, NANOG, PRDM14, and LIN28A) but with a notable lack of SOX2 expression (Figure 2C). Early mesoderm marker T was detected in hPGCLCs (Figure 2C), as in mouse early PGCs (Aramaki et al., 2013). Interestingly, expression of two endodermal genes, SOX17 and GATA4, was detected in hPGCLCs, embryonic hPGCs, and TCam-2, which are absent in the mouse germline. Notably, we identified CD38 expression in hPGCLCs/hPGCs and TCam-2, but not in hESCs or soma (Figures 2C and see also Figures 3A–3C). Overall, hPGCLCs indeed have germ cell characteristics consistent with hPGCs. Late germ cell markers, however, including DAZL, VASA, and MAEL, were only detected in hPGCs (Figure 2C). TCam-2 gene expression was similar to hPGCLCs, albeit with lower expression levels of NANOS3, ITGB3, and T and upregulation of a few somatic genes, e.g., HAND1 and RUNX1. Immunofluorescence analysis validated the expression of BLIMP1, TFAP2C, and OCT4 in hPGCLCs/hPGCs and TCam-2 (Figures 2E–2H). Interestingly, PRDM14 showed nuclear localization in the majority of hPGCLCs but was predominantly enriched in the cytoplasm of hPGCs (Figure 2F). Importantly, although SOX2 was undetectable, there was significant expression of SOX17 in hPGCLCs, hPGCs, and TCam-2 (Figures 2G and 2H).


SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

CD38 Expression in Human Germ-Cell-Related Cells and Epigenetic Changes in hPGCLCs(A) FACS analysis of NANOS3-mCherry and CD38 on WIS2-NANOS3-mCherry cell line cultured in 4i medium and on day 4 and 5 embryoids following hPGCLC induction. Ratios of CD38 low and high expression in the NANOS3-mCherry-positive cells are indicated.(B) FACS histogram of CD38 low and high populations in TCam-2.(C) FACS analysis of CD38 and TNAP on genital ridges isolated from a week 6 human embryo.(D) Expression analysis by RT-qPCR for FACS-sorted TNAP-positive 4i hESCs (TNAP+ hESC) and CD38 low or high/NANOS3-mCherry day 5 hPGCLCs. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(E and F) Immunofluorescence analysis for 5hmC (E) and TET1 (F) on day 4 embryoids cryosection. OCT4 or BLIMP1 were used to identify hPGCLCs (highlighted). Scale bars, 50 μm.(G) Quantification of immunofluorescence intensity of various epigenetic marks/modifiers in hPGCLCs and somatic neighbors in day 1–4 embryoids (see also Figures S3A–S3C). For UHRF1, only KI-67-positive (proliferating) cells were used for quantification. Numbers below each box denotes number of cells analyzed. Black central line represents the median, boxes and whiskers represent the 25th and 75th, and 2.5th and 97.5th percentiles, respectively. Wilcoxon signed-rank test was used to test for statistical significance. #p < 0.05; ∗p < 0.0001.
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fig3: CD38 Expression in Human Germ-Cell-Related Cells and Epigenetic Changes in hPGCLCs(A) FACS analysis of NANOS3-mCherry and CD38 on WIS2-NANOS3-mCherry cell line cultured in 4i medium and on day 4 and 5 embryoids following hPGCLC induction. Ratios of CD38 low and high expression in the NANOS3-mCherry-positive cells are indicated.(B) FACS histogram of CD38 low and high populations in TCam-2.(C) FACS analysis of CD38 and TNAP on genital ridges isolated from a week 6 human embryo.(D) Expression analysis by RT-qPCR for FACS-sorted TNAP-positive 4i hESCs (TNAP+ hESC) and CD38 low or high/NANOS3-mCherry day 5 hPGCLCs. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.(E and F) Immunofluorescence analysis for 5hmC (E) and TET1 (F) on day 4 embryoids cryosection. OCT4 or BLIMP1 were used to identify hPGCLCs (highlighted). Scale bars, 50 μm.(G) Quantification of immunofluorescence intensity of various epigenetic marks/modifiers in hPGCLCs and somatic neighbors in day 1–4 embryoids (see also Figures S3A–S3C). For UHRF1, only KI-67-positive (proliferating) cells were used for quantification. Numbers below each box denotes number of cells analyzed. Black central line represents the median, boxes and whiskers represent the 25th and 75th, and 2.5th and 97.5th percentiles, respectively. Wilcoxon signed-rank test was used to test for statistical significance. #p < 0.05; ∗p < 0.0001.
Mentions: A heat map of mRNA expression revealed that hPGCLCs and gonadal hPGCs shared expression of early PGCs (BLIMP1, TFAP2C, DND1, NANOS3, UTF1, ITGB3, and KIT) and pluripotency genes (TNAP, OCT4, NANOG, PRDM14, and LIN28A) but with a notable lack of SOX2 expression (Figure 2C). Early mesoderm marker T was detected in hPGCLCs (Figure 2C), as in mouse early PGCs (Aramaki et al., 2013). Interestingly, expression of two endodermal genes, SOX17 and GATA4, was detected in hPGCLCs, embryonic hPGCs, and TCam-2, which are absent in the mouse germline. Notably, we identified CD38 expression in hPGCLCs/hPGCs and TCam-2, but not in hESCs or soma (Figures 2C and see also Figures 3A–3C). Overall, hPGCLCs indeed have germ cell characteristics consistent with hPGCs. Late germ cell markers, however, including DAZL, VASA, and MAEL, were only detected in hPGCs (Figure 2C). TCam-2 gene expression was similar to hPGCLCs, albeit with lower expression levels of NANOS3, ITGB3, and T and upregulation of a few somatic genes, e.g., HAND1 and RUNX1. Immunofluorescence analysis validated the expression of BLIMP1, TFAP2C, and OCT4 in hPGCLCs/hPGCs and TCam-2 (Figures 2E–2H). Interestingly, PRDM14 showed nuclear localization in the majority of hPGCLCs but was predominantly enriched in the cytoplasm of hPGCs (Figure 2F). Importantly, although SOX2 was undetectable, there was significant expression of SOX17 in hPGCLCs, hPGCs, and TCam-2 (Figures 2G and 2H).

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Related in: MedlinePlus